Novel method for preventing or treating m tuberculosis infection

ABSTRACT

The present invention is directed to methods of preventing reactivation of active and latent  M. tuberculosis  infections by administering a pharmaceutical composition comprising a nucleic acid encoding a Mtb72f fusion protein, or a Mtb72f fusion protein or an immunogenic fragment thereof, for example together with an adjuvant. The Mtb72f nucleic acid or fusion protein can be administered with one or more chemotherapeutic agents effective against a  M. tuberculosis  infection. The methods also provide for shortening the time course of a chemotherapeutic regimen against a  M. tuberculosis  infection.

FIELD OF THE INVENTION

The present invention relates to methods of preventing or treatingreactivation of a M. tuberculosis infection in a mammal and to methodsof shortening the time course of chemotherapy against a M. tuberculosisinfection.

BACKGROUND OF THE INVENTION

Tuberculosis is a chronic infectious disease caused by infection with M.tuberculosis and other Mycobacterium species. It is a major disease indeveloping countries, as well as an increasing problem in developedareas of the world, with about 8 million new cases and 3 million deathseach year. Although the infection may be asymptomatic for a considerableperiod of time, the disease is most commonly manifested as an acuteinflammation of the lungs, resulting in fever and a nonproductive cough.If untreated, serious complications and death typically result.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behavior is difficult to monitor.Some patients do not complete the course of treatment, which can lead toineffective treatment and the development of drug resistance. Even if afull course of treatment is completed, infection with M. tuberculosis isnot eradicated from the infected individual but remains as a latentinfection that can be reactivated.

In order to control the spread of tuberculosis, effective vaccinationand accurate early diagnosis of the disease are of utmost importance.Currently, vaccination with live bacteria is the most efficient methodfor inducing protective immunity. The most common mycobacterium employedfor this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strainof M. bovis. However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public with this agent.

Diagnosis of tuberculosis is commonly achieved using a skin test, whichinvolves intradermal exposure to tuberculin PPD (protein-purifiedderivative). Antigen-specific T cell responses result in measurableinduration at the injection site by 48-72 hours after injection, whichindicates exposure to mycobacterial antigens. Sensitivity andspecificity have, however, been a problem with this test, andindividuals vaccinated with BCG cannot be distinguished from infectedindividuals.

While macrophages have been shown to act as the principal effectors ofMycobacterium immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection againstMycobacterium infection is illustrated by the frequent occurrence ofMycobacterium infection in AIDS patients, due to the depletion of CD4⁺ Tcells associated with human immunodeficiency virus (HIV) infection.Mycobacterium-reactive CD4⁺ T cells have been shown to be potentproducers of γ-interferon (IFN-γ), which, in turn, has been shown totrigger the anti-mycobacterial effects of macrophages in mice. While therole of IFN-γ in humans is less clear, studies have shown that1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-γ ortumor necrosis factor-alpha, activates human macrophages to inhibit M.tuberculosis infection. Furthermore, it is known that IFN-γ stimulateshuman macrophages to make 1,25-dihydroxy-vitamin D3. Similarly,interleukin-12 (IL-12) has been shown to play a role in stimulatingresistance to M. tuberculosis infection. For a review of the immunologyof M. tuberculosis infection, see Chan & Kaufmann, Tuberculosis:Pathogenesis, Protection and Control (Bloom ed., 1994), Tuberculosis(2nd ed., Rom and Garay, eds., 2003), and Harrison's Principles ofInternal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, etal., eds., 2005).

There remains a need for effective treatment strategies to preventreactivation of Mycobacterium tuberculosis infections, from both activeand latent infections. This invention fulfills this and other needs.

DESCRIPTION OF THE LISTED SEQUENCES

SEQ ID No:1: Mtb72f with N-terminal 6 His tag (DNA)

SEQ ID No:2: Mtb72f with N-terminal 6 His tag (protein)

SEQ ID No:3: M72 (variant of Mtb72f) with N-terminal 2 His insertion(DNA)

SEQ ID No:4: M72 (variant of Mtb72f) with N-terminal 2-His insertion(protein)

SEQ ID No:5: Mtb72f without N-terminal His insertion (DNA)

SEQ ID No:6: Mtb72f without N-terminal His insertion (protein)

BRIEF SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprising aMtb72f fusion protein or an immunogenic fragment thereof from aMycobacterium species of the tuberculosis complex, for example togetherwith one or more adjuvants, including AS01B and AS02A.

The present invention is based, in part, on the inventors' discoverythat administration of a Mtb72f fusion protein or immunogenic fragmentthereof eg together with one or more adjuvants or a nucleic acidencoding a Mtb72f fusion protein or immunogenic fragment thereof canprevent or treat reactivation of an active or inactive M. tuberculosisinfection. In a preferred embodiment, a Mtb72f fusion protein or nucleicacid is administrated with one or more chemotherapeutic agents effectiveagainst a M. tuberculosis infection.

In one aspect, the compositions are employed in methods for preventingor treating tuberculosis reactivation in a subject, the methodcomprising the step of administering to a mammal already infected withMycobacterium tuberculosis an immunologically effective amount of apharmaceutical composition comprising a Mtb72f fusion protein or animmunogenic fragment thereof from a Mycobacterium species of thetuberculosis complex and an adjuvant, wherein the Mtb72f fusion proteininduces an immune response against M. tuberculosis, thereby preventingor treating tuberculosis reactivation.

In another aspect, the compositions are employed in methods forpreventing tuberculosis reactivation in a subject, the method comprisingthe step of administering to a mammal already infected withMycobacterium tuberculosis an immunologically effective amount of apharmaceutical composition comprising a nucleic acid encoding a Mtb72ffusion protein or an immunogenic fragment thereof from a Mycobacteriumspecies of the tuberculosis complex, wherein the expressed Mtb72f fusionprotein induces an immune response against M. tuberculosis, therebypreventing or treating tuberculosis reactivation.

In another aspect, the compositions are employed in methods for reducingthe time course of chemotherapy against a M. tuberculosis infection, themethod comprising administering to a mammal already infected withMycobacterium tuberculosis one or more chemotherapeutic agents effectiveagainst a M. tuberculosis infection and an immunologically effectiveamount of a pharmaceutical composition comprising a Mtb72f fusionprotein or an immunogenic fragment thereof from a Mycobacterium speciesof the tuberculosis complex and an adjuvant, wherein said Mtb72f fusionprotein or immunogenic fragment thereof induces an immune responseagainst M. tuberculosis, thereby allowing for reducing the time courseof chemotherapy against a M. tuberculosis infection. By shortening thetime course of chemotherapy against a M. tuberculosis infection, thepresent methods are also effective in enhancing the compliance of anindividual being treated for a M. tuberculosis infection in completingan entire course of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graphic representation of the M. tuberculosisreactivation model in Swiss Webster mice (SWR/J). The figure showstimepoints for infection, chemotherapy treatment (50 mg rifampin/85 mgisoniazide per Liter of drinking water), immunizations and enumerationof bacterial load/colony forming units (CFU).

FIG. 2 shows IgG1 and IgG2a antibody responses immune responses in M.tuberculosis infected SWR/J mic treated with chemostherapy and then eimmunized with Mtb72f. Mice were left untreated, treated withchemotherapy (50 mg rifampin/85 mg isoniazide per Liter of drinkingwater) or treated with chemotherapy and immunized three timesintra-muscularly with 8 μg per dose of Mtb72f formulated withoutadjuvant. (FIG. 2A, FIG. 2B, FIG. 2C) Ten days after the lastimmunization the mice were bled and sera tested for anti-Mtb72f antibodyresponse for both IgG1 (red) and IgG2a (black) isotopes by ELISA.

FIG. 3 shows IgG1 and IgG2a antibody responses immune responses in M.tuberculosis infected SWR/J mice treated with chemotherapy and thenimmunized with Mtb72f. Mice were left untreated, treated withchemotherapy (50 mg rifampin/85 mg isoniazide per Liter of drinkingwater) or treated with chemotherapy and immunized three timesintra-muscularly with 8 μg per dose of Mtb72f formulated with theadjuvant AS01B. (FIG. 3A, FIG. 3B) Ten days after the last immunizationthe mice were bled and sera tested for anti-Mtb72f antibody response forboth IgG1 (red) and IgG2a (black) isotopes by ELISA.

FIG. 4 shows interferon-gamma (IFN-γ) responses in M. tuberculosisinfected SWR/J mice treated with chemotherapy and then immunized withMtb72f. Spleen cells were obtained from mice at varying timepoints andstimulated in vitro for three days with 10 μg/ml of either rMtb72f (FIG.4B) or the components (Mtb32_(C) and Mtb39) as indicated. (FIG. 4D, FIG.4F) As controls, splenocyte cultures were also stimulated with eitherPPD (3 μg/ml) (FIG. 4E), BCG Lysate (10 μg/ml) (FIG. 4G), conA (3μg/ml)(FIG. 4C) or medium alone (FIG. 4A). IFN-γ production was subsequentlymeasured by ELISA.

FIG. 5 shows IFN-γ responses in M. tuberculosis infected SWR/J micetreated with chemotherapy and then immunized with Mtb72f. Spleen cellswere obtained from mice at varying timepoints and stimulated in vitrofor three days with 10 μg/ml of either rMtb72f (FIG. 5B) or thecomponents (Mtb32_(C) and Mtb39) as indicated (FIG. 5D, FIG. 5F). Ascontrols, splenocyte cultures were also stimulated with either PPD (3μg/ml) (FIG. 5E), BCG Lysate (10 μg/ml) (FIG. 5G), conA (3 μg/ml) (FIG.5C) or medium alone (FIG. 5A). IFN-γ production was subsequentlymeasured by ELISA.

FIG. 6 (FIG. 6A.1-FIG. 6A.4; FIG. 6B1-FIG. 6B.4) shows CD4+ T cell andIFN-γ cytokine responses in M. tuberculosis infected SWR/J mice treatedwith chemotherapy and then immunized with Mtb72f. Spleen cells wereobtained from mice at varying timepoints and stimulated in vitroovernight with 10 μg/ml of rMtb72f. The cells were then stained for CD4and IFN-γ. As a control, splenocyte cultures were also stimulated withmedium alone. CD4+T cell specific IFN-γ+ production was subsequentlymeasured by intracellular cytokine staining (ICS).

FIG. 7 shows a tabular summary of the values of CD4+ and CD8+ T cellspecific IFN-γ+ production at Day 120 after Mtb infection. Spleen cellswere obtained from groups of mice left untreated, treated with 30, 60 or90 days of combination chemotherapy, or treated with combinationchemotherapy as an adjunct to the Mtb72f vaccine. Splenocytes werestimulated in vitro overnight with 10 μg/ml of rMtb72f. The cells werethen stained for CD4, CD8 or IFN-γ. As a control, splenocyte cultureswere also stimulated with medium alone. CD4+ and CD8+ T cell specificIFN-γ+ production was subsequently measured by intracellular cytokinestaining.

FIG. 8 shows survival of M. tuberculosis infected SWR/J mice treatedwith chemotherapy and then immunized with Mtb72f. Mice were infected viaaerosol with 50-100 CFU of MtbH37Rv and chemotherapy (50 mg rifampin/85mg isoniazide per Liter of drinking water) was started in a subset ofmice thirty days later. Chemotherapy was continued for 60 days. Half ofthose mice receiving chemotherapy were immunized three timesintra-muscularly with 8 μg per dose of Mtb72f formulated with theadjuvant AS01B.

FIG. 9 shows survival of M. tuberculosis infected SWR/J mice treatedwith chemotherapy and then immunized with Mtb72f. Mice were infected viaaerosol with 50-100 CFU of MtbH37Rv and chemotherapy (50 mg rifampin/85mg isoniazide per Liter of drinking water) was started in a subset ofmice thirty days later. Chemotherapy was continued for 30, 60 or 90 daysin separate subsets of mice. Half of those mice receiving chemotherapywere immunized three times intra-muscularly with 8 μg per dose of Mtb72fformulated with the adjuvant AS01B.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention relates to compositions comprising Mtb72f nucleicacids or fusion proteins and an adjuvant useful for treating,preventing, or delaying reactivation of an active or inactive (i.e.,latent) Mycobacterium infection, and methods for their use. Morespecifically, the compositions of the present invention comprise Mtb72ffusion polypeptides or immunogenic fragments thereof or nucleic acidsencoding Mtb72f fusion polypeptides or immunogenic fragments thereofhaving components from a Mycobacterium species of the tuberculosiscomplex, e.g., a species such as M. tuberculosis, M. bovis, or M.africanum, or a Mycobacterium species that is environmental oropportunistic and that causes opportunistic infections such as lunginfections in immune compromised hosts (e.g., patients with AIDS), e.g.,BCG, M. avium, M. intracellulare, M. celatum, M. genavense, M.haemophilum, M. kansasii, M. simiae, M. vaccae, M. fortuitum, and M.scrofulaceum (see, e.g., Harrison's Principles of Internal Medicine,Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005). Theinventors of the present application surprisingly discovered thatcompositions comprising Mtb72f fusion polypeptides or nucleic acidsencoding Mtb72f fusion polypeptides, or immunogenic fragments thereof,are useful in treating, preventing or delaying reactivation of a M.tuberulosis infection. In a preferred embodiment, a Mtb72f fusionpolypeptide or nucleic acid is administered with one or morechemotherapeutic agents. These compositions, polypeptides, and thenucleic acids that encode them are therefore useful for eliciting animmune response in mammals that is protective against reactivation ofdisease symptoms.

The Mtb72f nucleic acids and fusion polypeptides of the presentinvention can further comprise other components designed to enhancetheir antigenicity or to improve these antigens in other aspects. Forexample, improved isolation of the fusion polypeptide antigens may befacilitated through the addition of a stretch of histidine residuestowards one end of the antigen. The compositions, polypeptides, andnucleic acids of the invention can comprise additional copies ofantigens, or additional heterologous polypeptides from Mycobacteriumsp., such as MTB8.4 antigen, MTB9.8 antigen, MTB9.9 antigen, MTB40antigen, MTB41 antigen, ESAT-6 antigen, MTB85 complex antigen,α-crystalline antigen, or NS1 antigen. Alternatively or an addition thecompositions, polypeptides, and nucleic acids of the invention cancomprise additional copies of other antigens from Mycobacterium sp.,such as Ag85B or MTCC#2. The compositions, polypeptides, and nucleicacids of the invention can also comprise additional polypeptides fromother sources. For example, the compositions and fusion proteins of theinvention can include polypeptides or nucleic acids encodingpolypeptides, wherein the polypeptide enhances expression of theantigen, e.g., NS1, an influenza virus protein (see, e.g. WO99/40188 andWO93/04175). The nucleic acids of the invention can be engineered basedon codon preference in a species of choice, e.g., humans.

The Mtb72f fusion protein compositions usually comprise one or moreadjuvants, e.g., AS01B (monophosphoryl lipid A (MPL) and QS21 in aliposome formulation; see, U.S. Patent Publication No. 2003/0143240);AS2A (3D-MPL and QS21 and an oil in water emulsion; see, Bojang, et al.,Lancet (2001) 358:1927); , ENHANZYN (Detox); 3D-MPL; saponins includingQuil A and its components eg QS21 and saponin mimetics; CWS; TDM; AGP;immunostimulatory oligonucleotides eg CPG; Leif; and derivativesthereof. In a preferred embodiment, a Mtb72f fusion polypeptide isadministered with one or more adjuvants selected from the groupconsisting of 3D-MPL and QS21 in a liposome formulation eg AS01B and MPLand QS21 and an oil in water emulsion (eg AS02A). Adjuvants AS01B andAS02A are further described in Pichyangkul, et al., Vaccine (2004)22:3831-40.

When delivering the Mtb72f antigen as a nucleic acid, it can bedelivered, for example, in a viral vector (i.e., an adenovirus vector),or in a mutant bacterium host cell (i.e., a mutant, avirulentMycobacterium, Lactobacillus or Bacillus host cell including BacillusCalmette-Guerin (BCG) and Lactococcus lactis).

In one aspect, the compositions are employed in methods for preventingor treating tuberculosis reactivation in a subject, the methodcomprising the step of administering to a mammal already infected withMycobacterium tuberculosis an immunologically effective amount of apharmaceutical composition comprising a Mtb72f fusion protein or animmunogenic fragment thereof from a Mycobacterium species of thetuberculosis complex and an adjuvant, wherein the Mtb72f fusion proteininduces an immune response against M. tuberculosis, thereby preventingtuberculosis reactivation. By practicing the methods of the presentinvention, reactivation of a M. tuberculosis infection can be delayed(for example by a period of months, years or indefinitely).

In one aspect, the compositions are employed in methods for preventingor treating tuberculosis reactivation in a subject, the methodcomprising the step of administering to a mammal already infected withMycobacterium tuberculosis an immunologically effective amount of apharmaceutical composition comprising a nucleic acid encoding a Mtb72ffusion protein or an immunogenic fragment thereof from a Mycobacteriumspecies of the tuberculosis complex, wherein the expressed Mtb72f fusionprotein induces an immune response against M. tuberculosis, therebypreventing tuberculosis reactivation.

In one embodiment, the Mtb72f nucleic acid or fusion protein isadministered to an individual with an active M. tuberculosis infection.In one embodiment, the Mtb72f nucleic acid or fusion protein isadministered to an individual with an inactive or latent M. tuberculosisinfection. In one embodiment, the Mtb72f nucleic acid or fusion proteinis administered to an individual infected with a multi-drug resistantstrain of M. tuberculosis. In one embodiment, the Mtb72f nucleic acid orfusion protein is administered to an individual who was previouslyimmunized with Bacillus Calmette-Guerin (BCG).

In some embodiments, the Mtb72f nucleic acid or fusion protein isadministered with one or more chemotherapeutic agents effective againsta M. tuberculosis infection. Examples of such chemotherapeutic agentsinclude, but are not limited to, amikacin, aminosalicylic acid,capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin,pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin),streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin andfluoroquinolones. Such chemotherapy is determined by the judgment of thetreating physician using preferred drug combinations. “First-line”chemotherapeutic agents used to treat a M. tuberculosis infection thatis not drug resistant include isoniazid, rifampin, ethambutol,streptomycin and pyrazinamide. “Second-line” chemotherapeutic agentsused to treat a M. tuberculosis infection that has demonstrated drugresistance to one or more “first-line” drugs include ofloxacin,ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin,kanamycin and capreomycin.

The Mtb72f nucleic acid or fusion protein can be administered before,concurrently with, or after administration of the one or morechemotherapeutic agents effective against a M. tuberculosis infection.In one embodiment, the Mtb72f nucleic acid or fusion protein isadministered about 2 weeks after commencing administration of one ormore chemotherapeutic agents. The one or more chemotherapeutic agentsare generally administered over a period of time, for example, for about1, 2, 3, or 4 weeks, 2, 3, 4, 5, 6 or 8 months, 1 year or longer.

In certain embodiments, the effect of an Mtb72f nucleic acid or fusionprotein is enhanced by administration with Bacillus Calmette-Guerin(BCG).

In some embodiments, a priming or first administration of a Mtb72fnucleic acid or fusion polypeptide is followed by one or more “boosting”or subsequent administrations of a Mtb72f nucleic acid or fusionpolypeptide (“prime and boost” method). For instance, a firstadministration with a Mtb72f nucleic acid or fusion polypeptide isfollowed by one or more subsequent administrations of a Mtb72f nucleicacid or fusion protein. In one embodiment, a first administration with aMtb72f nucleic acid or fusion polypeptide is followed by one or moresubsequent administrations of a Mtb72f fusion polypeptide. In oneembodiment, a first administration with a Mtb72f nucleic acid or fusionpolypeptide is followed by one or more subsequent administrations of aMtb72f nucleic acid. Usually the first or “priming” administration andthe second or “boosting” administration are given about 2-12 weeksapart, or up to 4-6 months apart. Subsequent “booster” administrationsare given about 6 months apart, or as long as 1, 2, 3, 4 or 5 yearsapart. Conventional booster treatment (e.g., a protein primingadministration followed by a protein boosting administration) is alsouseful in preventing or treating against M. tuberculosis reactivation.

In another aspect, the compositions are employed in methods for reducingor shortening the time course of chemotherapy against a M. tuberculosisinfection, the method comprising administering to a mammal alreadyinfected with Mycobacterium tuberculosis one or more chemotherapeuticagents effective against a M. tuberculosis infection and animmunologically effective amount of a pharmaceutical compositioncomprising a Mtb72f fusion polypeptide or an immunogenic fragmentthereof from a Mycobacterium species of the tuberculosis complex and anadjuvant, wherein said Mtb72f fusion polypeptide induces an immuneresponse against M. tuberculosis, thereby allowing for reducing orshortening the time course of chemotherapy against a M. tuberculosisinfection. Usually, administration of a Mtb72f nucleic acid or fusionpolypeptide will allow effective chemotherapeutic treatment against a M.tuberculosis infection within 6 months, 5 months, 4 months, 3 months, orless.

The Mtb72f compositions are usually administered to humans, but areeffective in other mammals including domestic mammals (i.e., dogs, cats,rabbits, rats, mice, guinea pigs, hamsters, chinchillas) andagricultural mammals (i.e., cows, pigs, sheep, goats, horses).

In its most general respect, a Mtb72f fusion protein according to theinvention is a protein comprising at least an immunogenic fragment ofeach of the 3 antigens Ra12-TbH9-Ra35.

In the nomenclature of the application, Ra35 refers to the N-terminus ofMtb32A (Ra35FL), comprising at least about the first 205 amino acids ofMtb32A from M. tuberculosis, the nucleotide and amino acid sequence ofwhich is disclosed in FIG. 4 of U.S. patent application Ser. No.09/597,796, or the corresponding region from another Mycobacteriumspecies. Most typically, Ra35 refers to the portion of SEQ ID No: 2disclosed in the present application corresponding to residues 535-729.Alternatively it refers to a variant on Ra35 in which the amino acid Sercorresponding to 710 in SEQ ID No: 2 is replaced with Ala.

Ra12 refers to the C-terminus of Mtb32A (Ra35FL), comprising at leastabout the last 132 amino acids from MTB32A from M. tuberculosis, thesequence of which is disclosed as SEQ ID NO:4 (DNA) and SEQ ID NO:66(predicted amino acid sequence) in the U.S. patent application Ser. No.09/072,967, or the corresponding region from another Mycobacteriumspecies. Most typically, Ra12 refers to the portion of SEQ ID No: 2disclosed in the present application corresponding to residues 8-139.

Mtb39 (TbH9) refers to a sequence essentially that which is disclosed asSEQ ID NO:106 (cDNA full length) and SEQ ID NO:107 (protein full length)in the U.S. patent applications No. 08/658,800, No. 08/659,683, No.08/818,112, and No. 08/818,111 and in the WO97/09428 and WO97/09429applications. The sequence is also disclosed as SEQ ID NO:33 (DNA) andSEQ ID NO:91 (amino acid) in U.S. patent application Ser. No.09/056,559. Most typically, TbH9 refers to the portion of SEQ ID No: 2disclosed in the present application corresponding to residues 143-532.

The following provides sequences of some individual antigens used in thecompositions and fusion proteins of the invention:

Mtb32A (TbRa35FL or Ra35FL), the sequence of which is disclosed as SEQID NO:17 (cDNA) and SEQ ID NO:79 (protein) in the U.S. patentapplication Ser. No. 08/523,436, 08/523,435, No. 08/658,800, No.08/659,683, No. 08/818,112, No. 09/056,556, and No. 08/818,111 and inthe WO97/09428 and WO97/09429 applications, see also Skeiky et al.,Infection and Immunity 67:3998-4007 (1999);

The following provides sequences of some fusion proteins of theinvention:

TbH9-Ra35 (Mtb59F), the sequence of which is disclosed as SEQ ID NO:23(cDNA) and SEQ ID NO:24 (protein) in the U.S. patent application Ser.No. 09/287,849 and in the PCT/US99/07717 application;

Ra12-TbH9-Ra35 (Mtb72f), the sequence of which is disclosed as SEQ IDNO:1 or SEQ ID NO: 5 (DNA) and SEQ ID NO:2 or SEQ ID NO:6 (protein) inthe present application, as well as in U.S. patent application Ser. No.09/223,040, and in PCT/US99/07717. The sequences of SEQ ID NO: 1 and SEQID NO:2 include a His tag of 6 His residues.

M72 which is a mutant of Mtb72f in which the serine residue at aminoacid corresponding to position 710 in SEQ ID No: 2 has been changed toAla, (as well as 4 His residues having been removed from the His-tag atthe N terminus) the sequence of which is disclosed as SEQ ID No: 3 (DNA)and SEQ ID No: 4 (protein) in the present application. A variant onthese sequences in which the protein has a His tag of 6 His residues isdisclosed in U.S. patent application Ser. No. 09/597,796 and inPCT/US01/19959. By virtue of the replacement of Ser710 with Ala, M72 isbelieved to be more resistant to autolysis than Mtb72f.

The following provides sequences of some additional antigens used in thecompositions and fusion proteins of the invention:

Mtb8.4 (DPV), the sequence of which is disclosed as SEQ ID NO:101 (cDNA)and SEQ ID NO:102 (protein) in the U.S. patent application Ser. No.08/658,800, No. 08/659,683, No. 08/818,112 and No. 08/818,111 and in theWO97/09428 and WO97/09429 applications;Mtb9.8 (MSL), the sequence of which is disclosed as SEQ ID NO:12 (DNA),SEQ ID NO:109 (predicted amino acid sequence) and SEQ ID NO:110 to 124(peptides) in the U.S. patent application Ser. No. 08/859,381, No.08/858,998, No. 09/073,009 and No. 09/073,010 and in the PCT/US98/10407and PCT/US98/10514 applications;Mtb9.9A (MTI, also known as MTI-A), the sequence of which is disclosedas SEQ ID NO:3 and SEQ ID NO:4 (DNA) and SEQ ID NO:29 and SEQ ID NO:51to 66 (ORF peptide for MTI) in the U.S. patent application Ser. No.08/859,381, No. 08/858,998, No. 09/073,009 and v09/073,010 and in thePCT/US98/10407 and PCT/US98/10514 applications. Two other MTI variantsalso exist, called MTI-B and MTI-C;Mtb40 (HTCC#1), the sequence of which is disclosed as SEQ ID NO:137(cDNA) and 138 (predicted amino acid sequence) in the U.S. patentapplication Ser. No. 09/073,009 and No. 09/073,010 and in thePCT/US98/10407 and PCT/US98/10514 applications;Mtb41 (MTCC#2), the sequence of which is disclosed as SEQ ID NO:140(cDNA) and SEQ ID NO:142 (predicted amino acid sequence) in the U.S.patent application Ser. No. 09/073,009 and No. 09/073,010 and in thePCT/US98/10407 and PCT/US98/10514 applications;ESAT-6, the sequence of which is disclosed as SEQ ID NO:103 (DNA) andSEQ ID NO:104 (predicted amino acid sequence) in the U.S. patentapplication Ser. No. 09/072,967. The sequence of ESAT-6 is alsodisclosed in U.S. Pat. No. 5,955,077;α-crystalline antigen, the sequence of which is disclosed in Verbon etal., J. Bact. 174:1352-1359 (1992);85 complex antigen, the sequence of which is disclosed in Content etal., Infect. & Immunol. 59:3205-3212 (1991).

Each of the above sequences is also disclosed in Cole et al. Nature393:537 (1998) and can be found at, e.g., http://www.sanger.ac.uk andhttp:/www.pasteur.fr/mycdb/.

The above sequences are disclosed in U.S. patent application Ser. Nos.08/523,435, 08/523,436, 08/658,800, 08/659,683, 08/818,111, 08/818,112,08/942,341, 08/942,578, 08/858,998, 08/859,381, 09/056,556, 09/072,596,09/072,967, 09/073,009, 09/073,010, 09/223,040, 09/287,849 and in PCTpatent applications PCT/US98/10407, PCT/US98/10514, PCT/US99/03265,PCT/US99/03268, PCT/US99/07717, WO97/09428 and WO97/09429, WO98/16645,WO98/16646, each of which is herein incorporated by reference.

The antigens described herein include polymorphic variants andconservatively modified variations, as well as inter-strain andinterspecies Mycobacterium homologs. In addition, the antigens describedherein include subsequences or truncated sequences. The fusion proteinsmay also contain additional polypeptides, optionally heterologouspeptides from Mycobacterium or other sources. These antigens may bemodified, for example, by adding linker peptide sequences as describedbelow. These linker peptides may be inserted between one or morecomponents which make up each of the fusion proteins.

DEFINITIONS

The term “tuberculosis reactivation” refers to the later manifestationof disease symptoms in an individual that tests positive in a tuberculintest but does not have apparent disease symptoms. The individual isinfected with M. tuberculosis, and may or may not have previouslymanifested active disease symptoms that had been treated sufficiently tobring the tuberculosis into an inactive or latent state. Methods for theprevention or treatment of tuberculosis reactivation can be initiated inan individual manifesting active symptoms of disease, however.

“Primary tuberculosis” refers to clinical illness (manifestation ofdisease symptoms) directly following infection with M. tuberculosis.See, Harrison's Principles of Internal Medicine, Chapter 150, pp.953-966 (16th ed., Braunwald, et al., eds., 2005).

“Secondary tuberculosis” or “postprimary tuberculosis” refers to thereactivation of a dormant, inactive or latent M. tuberculosis infection.See, Harrison's Principles of Internal Medicine, supra.

An “active infection of M. tuberculosis” refers to a M. tuberculosisinfection with manifested disease symptoms.

An “inactive, dormant or latent infection of M. tuberculosis” refers toa M. tuberculosis infection without manifested disease symptoms.

A “drug resistant” M. tuberculosis infection refers to a M. tuberculosisinfection wherein the infecting strain is not held static or killed (isresistant to) one or more of so-called “front-line” chemotherapeuticagents effective in treating a M. tuberculosis infection (e.g.,isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide).

A “multi-drug resistant” M. tuberculosis infection refers to a M.tuberculosis infection wherein the infecting strain is resistant to twoor more of “front-line” chemotherapeutic agents effective in treating aM. tuberculosis infection.

A “chemotherapeutic agent effective in treating a M. tuberculosisinfection” refers to pharmacological agents known and used in the art totreat M. tuberculosis infections. Exemplified pharmacological agentsused to treat M. tuberculosis infections include, but are not limited toamikacin, aminosalicylic acid, capreomycin, cycloserine, ethambutol,ethionamide, isoniazid, kanamycin, pyrazinamide, rifamycins (i.e.,rifampin, rifapentine and rifabutin), streptomycin, ofloxacin,ciprofloxacin, clarithromycin, azithromycin and fluoroquinolones.“First-line” chemotherapeutic agents used to treat a M. tuberculosisinfection that is not drug resistant include isoniazid, rifampin,ethambutol, streptomycin and pyrazinamide. “Second-line”chemotherapeutic agents used to treat a M. tuberculosis infection thathas demonstrated drug resistance to one or more “first-line” drugsinclude ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid,cycloserine, amikacin, kanamycin and capreomycin. Such pharmacologicalagents are reviewed in Chapter 48 of Goodman and Gilman's ThePharmacological Basis of Therapeutics, Hardman and Limbird eds., 2001.

“FL” refers to full-length, i.e., a polypeptide that is the same lengthas the wild-type polypeptide.

“His tag” refers to a string of His residues, typically 6 residues thatare inserted at the N-terminus, usually immediately after the initiatingMet residue or else at the C-terminus They are usually heterologous tothe native sequence but are incorporated since they facilitate isolationby improving the protein binding to immobilised metal affinitychromatography resins (IMAC). Generally speaking the presence or absenceof a His tag is not of significance from the point of view of causing auseful immune response against the antigenic protein to be elicited. Incase an adverse immune reaction against the His tag itself is elicitedit is considered best to minimize the length of the His tag eg to 4 orless residues, in particular two residues.

The term “immunogenic fragment thereof” refers to a polypeptidecomprising an epitope that is recognized by cytotoxic T lymphocytes,helper T lymphocytes or B cells. Typically an immunogenic fragment ofMtb72f will be a polypeptide containing 500 or more amino acids eg 600or more amino acids eg 700 or more amino acids. The invention alsoembraces a plurality of fragments eg overlapping fragments whichtogether cover all or substantially all (eg 500 or more amino acids eg600 or more amino acids eg 700 or more amino acids) of the sequence of aMtb72F fusion protein.

The term “Mycobacterium species of the tuberculosis complex” includesthose species traditionally considered as causing the diseasetuberculosis, as well as Mycobacterium environmental and opportunisticspecies that cause tuberculosis and lung disease in immune compromisedpatients, such as patients with AIDS, e.g., M. tuberculosis, M. bovis,or M. africanum, BCG, M. avium, M. intracellulare, M. celatum, M.genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, M.fortuitum, and M. scrofulaceum (see, e.g., Harrison's Principles ofInternal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, etal., eds., 2005).

An adjuvant refers to the components in a vaccine or therapeuticcomposition that increase the specific immune response to the antigen(see, e.g., Edelman, AIDS Res. Hum Retroviruses 8:1409-1411 (1992)).Adjuvants induce immune responses of the Th1-type and Th-2 typeresponse. Th1-type cytokines (e.g., IFN-γ, IL-2, and IL-12) tend tofavor the induction of cell-mediated immune response to an administeredantigen, while Th-2 type cytokines (e.g., IL-4, IL-5, Il-6, IL-10 andTNF-β) tend to favor the induction of humoral immune responses.Adjuvants capable of preferential stimulation of a Th-1 cell-mediatedimmune response are described in WO 94/00153 and WO 95/17209.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule.

Accordingly, each silent variation of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

“Fusion polypeptide” or “fusion protein” refers to a protein having atleast two heterologous Mycobacterium sp. polypeptides covalently linked,either directly or via an amino acid linker. The polypeptides formingthe fusion protein are typically linked C-terminus to N-terminus,although they can also be linked C-terminus to C-terminus, N-terminus toN-terminus, or N-terminus to C-terminus. The polypeptides of the fusionprotein can be in any order. This term also refers to conservativelymodified variants, polymorphic variants, alleles, mutants, subsequences,and interspecies homologs of the antigens that make up the fusionprotein. Mycobacterium tuberculosis antigens are described in Cole etal., Nature 393:537 (1998), which discloses the entire Mycobacteriumtuberculosis genome. The complete sequence of Mycobacterium tuberculosiscan also be found at http://www.sanger.ac.uk and athttp://www.pasteur.fr/mycdb/ (MycDB). Antigens from other Mycobacteriumspecies that correspond to M. tuberculosis antigens can be identified,e.g., using sequence comparison algorithms, as described herein, orother methods known to those of skill in the art, e.g., hybridizationassays and antibody binding assays.

Exemplary Mtb72f fusion proteins of use in the present inventioninclude:

Proteins comprising residues 8-729 of the sequence of SEQ ID No: 2;Proteins comprising or consisting of the sequence of SEQ ID No: 2(=Mtb72f) optionally without the His tag forming residues 2-7 of saidsequence or with a His tag of different length;Fusion proteins comprising the sequence of SEQ ID No: 2 optionallywithout the His tag forming residues 2-7 of said sequence or with a Histag of different length (e.g. a protein comprising residues 8-729 of thesequence of SEQ ID No: 2) together with one or more M. tuberculosisantigens, for example one or more of the proteins listed in paragraphs[0045] to [0052] above, or an immunogenic fragment of any of them;Proteins comprising residues 4-725 of the sequence of SEQ ID No: 4(=M72);Proteins comprising or consisting of the sequence of SEQ ID No: 4 (=M72)optionally without the His tag forming residues 2-3 of said sequence orwith a His tag of different length;andFusion proteins comprising the sequence of SEQ ID No: 4 optionallywithout the His tag forming residues 2-3 of said sequence or with a Histag of different length (e.g. a protein comprising residues 4-725 of thesequence of SEQ ID No: 4) together with one or more M. tuberculosisantigens, for example one or more of the proteins listed in paragraphs[0045] to [0052] above, or an immunogenic fragment of any of them;

Exemplary immunogenic fragments of a Mtb72f fusion proteins of use inthe present invention include:

Proteins comprising or consisting of the sequence of TbH9-Ra35 (Mtb59F);or TbH9; or Ra35; or Ra12; andFusion proteins comprising said sequences together with one or more M.tuberculosis antigens, for example one or more of the proteins listed inparagraphs [0045] to [0052] above, or an immunogenic fragment of any ofthem.

Further exemplary immunogenic fragments of a Mtb72f fusion proteins ofuse in the present invention include:

Proteins comprising or consisting of the sequence of TbH9-Ra35 (Mtb59F)or Ra35 in which the position corresponding to Ser710 in SEQ ID No: 2has been changed to Ala; andFusion proteins comprising said sequences together with one or more M.tuberculosis antigens, for example one or more of the proteins listed inparagraphs [0045] to [0052] above, or an immunogenic fragment of any ofthem.

More specifically the Mtb72f is:

-   -   a polypeptide comprising residues 8-729 of SEQ ID NO:2; or    -   a polypeptide consisting of residues 1 and 8-729 of SEQ ID NO:2        optionally with a His tag inserted following the initial Met        residue; or    -   a polypeptide of SEQ ID NO:2; or    -   a polypeptide comprising residues 4-725 of SEQ ID NO:4; or    -   a polypeptide consisting of residues 1 and 4-725 of SEQ ID NO:4        optionally with a His tag inserted following the initial Met        residue.; or    -   a polypeptide of SEQ ID NO:4.; or    -   a polypeptide of SEQ ID NO:6.

Further exemplary Mtb72f fusion proteins and immunogenic fragmentsthereof include the proteins mentioned above in which the N- and/or theC-terminus have been shortened by eg 5 or 4 or 3 or 2 or 1 amino acidresidues.

Further exemplary Mtb72f fusion proteins and immunogenic fragmentsthereof include the proteins mentioned above in which up to 10% of theamino acids, eg up to 5% of the amino acids (eg up 10 eg up to 5) aminoacids have been replaced with conservative substitutions as definedherein.

Exemplary Mtb72f nucleic acids of use in the present invention includenucleic acids (e.g. DNA molecules) encoding the aforementioned exemplaryMtb72f fusion proteins and immunogenic fragments thereof. One set ofspecific DNA molecules that may be mentioned comprise nucleotides63-2228 of SEQ ID No: 1. Another set of specific DNA molecules that maybe mentioned comprise nucleotides 10-2175 of SEQ ID No: 3. Specific DNAmolecules that may be mentioned comprise or consist of SEQ ID No: 1 orSEQ ID No: 3 or SEQ ID No: 5.

The term “fused” refers to the covalent linkage between two polypeptidesin a fusion protein. The polypeptides are typically joined via a peptidebond, either directly to each other or via an amino acid linker.Optionally, the peptides can be joined via non-peptide covalent linkagesknown to those of skill in the art.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to fusion proteins can be selected to obtain only thosepolyclonal antibodies that are specifically immunoreactive with fusionprotein and not with individual components of the fusion proteins. Thisselection may be achieved by subtracting out antibodies that cross-reactwith the individual antigens. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) and UsingAntibodies: A Laboratory Manual (1998), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an individual antigen or a portion thereof) or maycomprise a variant of such a sequence. Polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not diminished, relative to a fusion polypeptidecomprising native antigens. Variants preferably exhibit at least about70% identity, more preferably at least about 80% identity and mostpreferably at least about 90% identity to a polynucleotide sequence thatencodes a native polypeptide or a portion thereof.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95% identity overa specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 25 to about 50 amino acids or nucleotidesin length, or optionally over a region that is 75-100 amino acids ornucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 25 to 500, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Polynucleotide Compositions

As used herein, the terms “DNA segment” and “polynucleotide” refer to aDNA molecule that has been isolated free of total genomic DNA of aparticular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the DNA segments ofthis invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

“Isolated,” as used herein, means that a polynucleotide is substantiallyaway from other coding sequences, and that the DNA segment does notcontain large portions of unrelated coding DNA, such as largechromosomal fragments or other functional genes or polypeptide codingregions. Of course, this refers to the DNA segment as originallyisolated, and does not exclude genes or coding regions later added tothe segment by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Mycobacterium antigen or a portion thereof) ormay comprise a variant, or a biological or antigenic functionalequivalent of such a sequence. Polynucleotide variants may contain oneor more substitutions, additions, deletions and/or insertions, asfurther described below, preferably such that the immunogenicity of theencoded polypeptide is not diminished, relative to a native tumorprotein. The effect on the immunogenicity of the encoded polypeptide maygenerally be assessed as described herein. The term “variants” alsoencompasses homologous genes of xenogenic origin.

In additional embodiments, the present invention provides isolatedpolynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative DNA segments withtotal lengths of about 10,000, about 5000, about 3000, about 2,000,about 1,000, about 500, about 200, about 100, about 50 base pairs inlength, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention, for example polynucleotides that are optimized forhuman and/or primate codon selection. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

Polynucleotide Identification and Characterization

Polynucleotides may be identified, prepared and/or manipulated using anyof a variety of well established techniques. For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for tumor-associated expression (i.e.,expression that is at least two fold greater in a tumor than in normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using a Synteni microarray(Palo Alto, Calif.) according to the manufacturer's instructions (andessentially as described by Schena et al., Proc. Natl. Acad. Sci. USA93:10614-10619 (1996) and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155 (1997)). Alternatively, polynucleotides may be amplifiedfrom cDNA prepared from cells expressing the proteins described herein,such as M. tuberculosis cells. Such polynucleotides may be amplified viapolymerase chain reaction (PCR). For this approach, sequence-specificprimers may be designed based on the sequences provided herein, and maybe purchased or synthesized.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., a M.tuberculosis cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual (2000)). Hybridizingcolonies or plaques are selected and expanded, and the DNA is isolatedfor further analysis. cDNA clones may be analyzed to determine theamount of additional sequence by, for example, PCR using a primer fromthe partial sequence and a primer from the vector. Restriction maps andpartial sequences may be generated to identify one or more overlappingclones. The complete sequence may then be determined using standardtechniques, which may involve generating a series of deletion clones.The resulting overlapping sequences can then be assembled into a singlecontiguous sequence. A full length cDNA molecule can be generated byligating suitable fragments, using well known techniques.

Alternatively, there are numerous amplification techniques for obtaininga full length coding sequence from a partial cDNA sequence. Within suchtechniques, amplification is generally performed via PCR. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers are preferably 22-30 nucleotides inlength, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al.,Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19 (1991)) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60 (1991)). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

Polynucleotide Expression in Host Cells

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Horn et al.,Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, theprotein itself may be produced using chemical methods to synthesize theamino acid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge et al., Science 269:202-204 (1995)) and automated synthesis maybe achieved, for example, using the ABI 431A Peptide Synthesizer (PerkinElmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, Proteins,Structures and Molecular Principles (1983)) or other comparabletechniques available in the art. The composition of the syntheticpeptides may be confirmed by amino acid analysis or sequencing (e.g.,the Edman degradation procedure). Additionally, the amino acid sequenceof a polypeptide, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (2000), and Ausubel et al.,Current Protocols in Molecular Biology (updated annually).

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, for example for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified may be used. Such vectors include,but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke &Schuster, J. Biol. Chem. 264:5503-5509(1989)); and the like. pGEX Vectors (Promega, Madison, Wis.) may also beused to express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987).

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw HillYearbook of Science and Technology pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk Proc. Natl. Acad. Sci.U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers, suchas the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells. Methods and protocols for workingwith adenovirus vectors are reviewed in Wold, Adenovirus Methods andProtocols, 1998. Additional references regarding use of adenovirusvectors can be found in Adenovirus: A Medical Dictionary, Bibliography,and Annotated Research Guide to Internet References, 2004.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-32 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-23 (1990)) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes et al., Methods Mol.Biol. 55:121-131 (1995)).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton etal., Serological Methods, a Laboratory Manual (1990) and Maddox et al.,J. Exp. Med. 158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porathet al., Prot. Exp. Purif. 3:263-281 (1992) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll et al., DNA Cell Biol. 12:441-453 (1993)).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

In Vivo Polynucleotide Delivery Techniques

In additional embodiments, genetic constructs comprising one or more ofthe polynucleotides of the invention are introduced into cells in vivo.This may be achieved using any of a variety or well known approaches,several of which are outlined below for the purpose of illustration.

1. Adenovirus

One of the preferred methods for in vivo delivery of one or more nucleicacid sequences involves the use of an adenovirus expression vector.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express a polynucleotide that has been clonedtherein in a sense or antisense orientation. Of course, in the contextof an antisense construct, expression does not require that the geneproduct be synthesized.

The expression vector comprises a genetically engineered form of anadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus &Horwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones & Shenk, 1978), the current adenovirus vectors, with the help of293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham & Prevec, 1991). In nature, adenovirus can package approximately105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providingcapacity for about 2 extra kB of DNA. Combined with the approximately5.5 kB of DNA that is replaceable in the E1 and E3 regions, the maximumcapacity of the current adenovirus vector is under 7.5 kB, or about 15%of the total length of the vector. More than 80% of the adenovirus viralgenome remains in the vector backbone and is the source of vector-bornecytotoxicity. Also, the replication deficiency of the E1-deleted virusis incomplete. For example, leakage of viral gene expression has beenobserved with the currently available vectors at high multiplicities ofinfection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus& Horwitz, 1992; Graham & Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet & Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz & Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

Adenovirs vectors may originate from human adenovirus. Alternativelythey may originate from adenovirus of other species eg chimpanzee whichmay have the advantage that the viral vectors are not neutralized byantibodies against human adenovirus circulating in many human subjects(see eg: Tatsis N et al (2005) Gene Ther. December 1; [Epub ahead ofprint]).

2. Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding oneor more oligonucleotide or polynucleotide sequences of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and packaging components is constructed (Mann etal., 1983). When a recombinant plasmid containing a cDNA, together withthe retroviral LTR and packaging sequences is introduced into this cellline (by calcium phosphate precipitation for example), the packagingsequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas & Rubenstein, 1988; Temin, 1986; Mann et al., 1983). Themedia containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells (Paskind etal., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

3. Adeno-Associated Viruses

AAV (Ridgeway, 1988; Hermonat & Muzycska, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replications is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterized. AAV has a single-stranded linearDNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to forman icosahedral virion of 20 to 24 nm in diameter (Muzyczka & McLaughlin,1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two openreading frames and is flanked by two ITRs. There are two major genes inthe AAV genome: rep and cap. The rep gene codes for proteins responsiblefor viral replications, whereas cap codes for capsid protein VP1-3. EachITR forms a T-shaped hairpin structure. These terminal repeats are theonly essential cis components of the AAV for chromosomal integration.Therefore, the AAV can be used as a vector with all viral codingsequences removed and replaced by the cassette of genes for delivery.Three viral promoters have been identified and named p5, p19, and p40,according to their map position. Transcription from p5 and p19 resultsin production of rep proteins, and transcription from p40 produces thecapsid proteins (Hermonat & Muzyczka, 1984).

There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering theantisense constructs of the present invention.

AAV is also a good choice of delivery vehicles due to its safety. Thereis a relatively complicated rescue mechanism: not only wild typeadenovirus but also AAV genes are required to mobilize rAAV. Likewise,AAV is not pathogenic and not associated with any disease. The removalof viral coding sequences minimizes immune reactions to viral geneexpression, and therefore, rAAV does not evoke an inflammatory response.

4. Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention for the delivery of oligonucleotide or polynucleotidesequences to a host cell. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio virusesand herpes viruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

5. Non-Viral Vectors

In order to effect expression of the oligonucleotide or polynucleotidesequences of the present invention, the expression construct must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cells lines, or in vivo or exvivo, as in the treatment of certain disease states. As described above,one preferred mechanism for delivery is via viral infection where theexpression construct is encapsulated in an infectious viral particle.

Once the expression construct has been delivered into the cell thenucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty & Reshef (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

Polypeptide Compositions

The present invention, in other aspects, provides polypeptidecompositions. Generally, a polypeptide of the invention will be anisolated polypeptide (or an epitope, variant, or active fragmentthereof) derived from a mammalian species. Preferably, the polypeptideis encoded by a polynucleotide sequence disclosed herein or a sequencewhich hybridizes under moderately stringent conditions to apolynucleotide sequence disclosed herein. Alternatively, the polypeptidemay be defined as a polypeptide which comprises a contiguous amino acidsequence from an amino acid sequence disclosed herein, or whichpolypeptide comprises an entire amino acid sequence disclosed herein.

Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well known techniques. An immunogenic portion of aMycobacterium sp. protein is a portion that reacts with such antiseraand/or T-cells at a level that is not substantially less than thereactivity of the full length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Such immunogenic portions may react withinsuch assays at a level that is similar to or greater than the reactivityof the full length polypeptide. Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow & Lane, Antibodies: A Laboratory Manual (1988)and Using Antibodies: A Laboratory Manual (1998). For example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵I-labeled Protein A.

Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

Polypeptides of the invention, immunogenic fragments thereof, and othervariants having less than about 100 amino acids, and generally less thanabout 50 amino acids, may also be generated by synthetic means, usingtechniques well known to those of ordinary skill in the art. Forexample, such polypeptides may be synthesized using any of thecommercially available solid-phase techniques, such as the Merrifieldsolid-phase synthesis method, where amino acids are sequentially addedto a growing amino acid chain. See Merrifield, J. Am. Chem. Soc.85:2149-2146 (1963). Equipment for automated synthesis of polypeptidesis commercially available from suppliers such as Perkin Elmer/AppliedBioSystems Division (Foster City, Calif.), and may be operated accordingto the manufacturer's instructions.

Within certain specific embodiments, a polypeptide may be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length. Linker sequences are not required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided. Such proteins comprise a polypeptideas described herein together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a recallresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, e.g., Stoute et al., New Engl. J. Med.336:86-91 (1997)).

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292 (1986)). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798 (1992)). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

T Cells

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for a Mycobacterium antigen. Such cells may generally beprepared in vitro or ex vivo, using standard procedures. For example, Tcells may be isolated from bone marrow, peripheral blood, or a fractionof bone marrow or peripheral blood of a patient, using a commerciallyavailable cell separation system, such as the Isolex™ System, availablefrom Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). Alternatively, T cells may be derived from related orunrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with a polypeptide of the invention,polynucleotide encoding such a polypeptide, and/or an antigen presentingcell (APC) that expresses such a polypeptide. Such stimulation isperformed under conditions and for a time sufficient to permit thegeneration of T cells that are specific for the polypeptide. Preferably,the polypeptide or polynucleotide is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the inventionif the T cells specifically proliferate, secrete cytokines or killtarget cells coated with the polypeptide or expressing a gene encodingthe polypeptide. T cell specificity may be evaluated using any of avariety of standard techniques. For example, within a chromium releaseassay or proliferation assay, a stimulation index of more than two foldincrease in lysis and/or proliferation, compared to negative controls,indicates T cell specificity. Such assays may be performed, for example,as described in Chen et al., Cancer Res. 54:1065-1070 (1994)).Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a polypeptide of the invention (100 ng/ml-100μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days should result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1 (1998)). T cells that have been activated inresponse to a polypeptide, polynucleotide or polypeptide-expressing APCmay be CD4⁺ and/or CD8⁺. Protein-specific T cells may be expanded usingstandard techniques. Within preferred embodiments, the T cells arederived from a patient, a related donor or an unrelated donor, and areadministered to the patient following stimulation and expansion.

For therapeutic purposes, CD4+ or CD8+ T cells that proliferate inresponse to a polypeptide, polynucleotide or APC can be expanded innumber either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a polypeptide, or a short peptide corresponding toan immunogenic portion of such a polypeptide, with or without theaddition of T cell growth factors, such as interleukin-2, and/orstimulator cells that synthesize a polypeptide. Alternatively, one ormore T cells that proliferate in the presence of ar protein can beexpanded in number by cloning. Methods for cloning cells are well knownin the art, and include limiting dilution.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell, antibody, andchemotherapeutic compositions disclosed herein inpharmaceutically-acceptable solutions for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy.

It will also be understood that, if desired, the nucleic acid segment(e.g., RNA or DNA) that expresses a polypeptide as disclosed herein maybe administered in combination with other agents as well, such as, e.g.,other proteins or polypeptides or various pharmaceutically-activeagents, including chemotherapeutic agents effective against a M.tuberculosis infection. In fact, there is virtually no limit to othercomponents that may also be included, given that the additional agentsdo not cause a significant adverse effect upon contact with the targetcells or host tissues. The compositions may thus be delivered along withvarious other agents as required in the particular instance. Suchcompositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation. Typically, formulationscomprising a therapeutically effective amount deliver about 2 μg toabout 50 μg Mtb72f polypeptide per administration, more typically about5 μg to about 40 μg Mtb72f polypeptide per administration.

1. Oral Delivery

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.A syrup of elixir may contain the active compound sucrose as asweetening agent methyl and propylparabens as preservatives, a dye andflavoring, such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

Typically, these formulations usually contain between 2 μg to 50 μg ofMtb72f polypeptide. Naturally, the amount of active compound(s) in eachtherapeutically useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

2. Injectable Delivery

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as describedin U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

3. Nasal and Buccal Delivery

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, buccal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs eg via nasal and buccalaerosol sprays has been described e.g., in U.S. Pat. No. 5,756,353 andU.S. Pat. No. 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

4. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No.5,741,516, specifically incorporated herein by reference in itsentirety). Further, various methods of liposome and liposome likepreparations as potential drug carriers have been reviewed (Takakura,1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434;U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No.5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporatedherein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., 1990; Muller et al., 1990). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems.

Liposomes have been used effectively to introduce genes, drugs (Heath &Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta &Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987), enzymes(Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses (Faller &Baltimore, 1984), transcription factors and allosteric effectors(Nicolau & Gersonde, 1979) into a variety of cultured cell lines andanimals. In addition, several successful clinical trails examining theeffectiveness of liposome-mediated drug delivery have been completed(Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier et al.,1988). Furthermore, several studies suggest that the use of liposomes isnot associated with autoimmune responses, toxicity or gonadallocalization after systemic delivery (Mori & Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 um. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;Douglas et al., 1987). To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1 μm)should be designed using polymers able to be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet theserequirements are contemplated for use in the present invention. Suchparticles may be are easily made, as described (Couvreur et al., 1980;1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry etal., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated hereinby reference in its entirety).

Vaccines

In certain preferred embodiments of the present invention, vaccines areprovided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccinepreparation is generally described in, for example, Powell & Newman,eds., Vaccine Design (the subunit and adjuvant approach) (1995).Pharmaceutical compositions and vaccines within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive. For example, one or more immunogenic portions ofother tumor antigens may be present, either incorporated into a fusionpolypeptide or as a separate compound, within the composition orvaccine.

Illustrative vaccines may contain DNA encoding one or more of thepolypeptides as described above, such that the polypeptide is generatedin situ. As noted above, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems. Numerous gene delivery techniques are well known in the art,such as those described by Rolland, Crit. Rev. Therap. Drug CarrierSystems 15:143-198 (1998), and references cited therein. Appropriatenucleic acid expression systems contain the necessary DNA sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). Bacterial delivery systems involve the administration of abacterium host cell (for example, a Mycobacterium, Bacillus orLactobacillus strain, including Bacillus-Calmette-Guerrin or Lactococcuslactis) that expresses an immunogenic portion of the polypeptide on itscell surface or secretes such an epitope (see, for example, Ferreira, etal., An Acad Bras Cienc (2005) 77:113-124; and Raha, et al., ApplMicrobiol Biotechnol (2005) PubMedID 15635459). In a preferredembodiment, the DNA may be introduced using a viral expression system(e.g., vaccinia or other pox virus, retrovirus, or adenovirus), whichmay involve the use of a non-pathogenic (defective), replicationcompetent virus. Suitable systems are disclosed, for example, inFisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321 (1989);Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103 (1989); Flexner et al.,Vaccine 8:17-21 (1990); U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627 (1988);Rosenfeld et al., Science 252:431-434 (1991); Kolls et al., Proc. Natl.Acad. Sci. USA 91:215-219 (1994); Kass-Eisler et al., Proc. Natl. Acad.Sci. USA 90:11498-11502 (1993); Guzman et al., Circulation 88:2838-2848(1993); and Guzman et al., Cir. Res. 73:1202-1207 (1993). Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA may also be “naked,” as described,for example, in Ulmer et al., Science 259:1745-1749 (1993) and reviewedby Cohen, Science 259:1691-1692 (1993). The uptake of naked DNA may beincreased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells. It will be apparent that avaccine may comprise both a polynucleotide and a polypeptide component.Such vaccines may provide for an enhanced immune response.

It will be apparent that a vaccine may contain pharmaceuticallyacceptable salts of the polynucleotides and polypeptides providedherein. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases, including organic bases (e.g., salts of primary,secondary and tertiary amines and basic amino acids) and inorganic bases(e.g., sodium, potassium, lithium, ammonium, calcium and magnesiumsalts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the vaccine compositions of this invention, the typeof carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of immunostimulants may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium species or Mycobacterium derived proteins. For example,delipidated, deglycolipidated M. vaccae (“pVac”) can be used. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);AS01B, AS02A, AS15, AS-2 and derivatives thereof (GlaxoSmithKline,Philadelphia, Pa.); CWS, TDM, Leif, aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may alsobe used as adjuvants.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Janeway, et al., Immunobiology,5^(th) Edition, 2001.

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-O-deacylated monophosphoryl lipid A (3D-MPL), optionallywith an aluminum salt (see, for example, Ribi, et al., 1986, Immunologyand Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY,pp. 407-419; GB 2122204B; GB 2220211; and U.S. Pat. No. 4,912,094). Apreferred form of 3D-MPL is in the form of an emulsion having a smallparticle size less than 0 2 mm in diameter, and its method ofmanufacture is disclosed in WO 94/21292. Aqueous formulations comprisingmonophosphoryl lipid A and a surfactant have been described in WO98/43670. Exemplified preferred adjuvants include AS01B (MPL and QS21 ina liposome formulation), 3D-MPL and QS21 in a liposome formulation, AS2A(MPL and QS21 and an oil in water emulsion), 3D-MPL and QS21 and an oilin water emulsion, and AS15, available from GlaxoSmithKline. MPLadjuvants are available from GlaxoSmithKline, Seattle, Wash. (see U.S.Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).

CpG-containing oligonucleotides (in which the CpG dinucleotide isunmethylated) also induce a predominantly Th1 response. CpG is anabbreviation for cytosine-guanosine dinucleotide motifs present in DNA.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352 (1996). CpG when formulated into vaccines, isgenerally administered in free solution together with free antigen (WO96/02555; McCluskie and Davis, supra) or covalently conjugated to anantigen (WO 98/16247), or formulated with a carrier such as aluminiumhydroxide ((Hepatitis surface antigen) Davis et al. supra;Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95(26),15553-8). CpG is known in the art as being an adjuvant that can beadministered by both systemic and mucosal routes (WO 96/02555, EP468520, Davis et al., J. Immunol, 1998, 160(2):870-876; McCluskie andDavis, J. Immunol., 1998, 161(9):4463-6).

Another preferred adjuvant is a saponin or saponin mimetics orderivatives, preferably QS21 (Aquila Biopharmaceuticals Inc.,Framingham, Mass.), which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprise an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210. Additional saponin adjuvants of use in thepresent invention include QS7 (described in WO 96/33739 and WO 96/11711)and QS17 (described in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1).

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2, AS2′, AS2,″ SBAS-4, or SBAS6,available from GlaxoSmithKline, Rixensart, Belgium), Detox (Corixa,Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties.

Further example adjuvants include synthetic MPL and adjuvants based onShiga toxin B subunit (see WO2005/112991).

Any vaccine provided herein may be prepared using well known methodsthat result in a combination of antigen, immune response enhancer and asuitable carrier or excipient. The compositions described herein may beadministered as part of a sustained release formulation (i.e., aformulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438 (1996)) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see, e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets tumor cells. Deliveryvehicles include antigen presenting cells (APCs), such as dendriticcells, macrophages, B cells, monocytes and other cells that may beengineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau & Steinman, Nature 392:245-251(1998)) and have been shown to be effective as a physiological adjuvantfor eliciting prophylactic or therapeutic antitumor immunity (seeTimmerman & Levy, Ann. Rev. Med. 50:507-529 (1999)). In general,dendritic cells may be identified based on their typical shape (stellatein situ, with marked cytoplasmic processes (dendrites) visible invitro), their ability to take up, process and present antigens with highefficiency and their ability to activate naïve T cell responses.Dendritic cells may, of course, be engineered to express specificcell-surface receptors or ligands that are not commonly found ondendritic cells in vivo or ex vivo, and such modified dendritic cellsare contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600 (1998)).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding aprotein (or portion or other variant thereof) such that the polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a composition or vaccinecomprising such transfected cells may then be used for therapeuticpurposes, as described herein. Alternatively, a gene delivery vehiclethat targets a dendritic or other antigen presenting cell may beadministered to a patient, resulting in transfection that occurs invivo. In vivo and ex vivo transfection of dendritic cells, for example,may generally be performed using any methods known in the art, such asthose described in WO 97/24447, or the gene gun approach described byMahvi et al., Immunology and Cell Biology 75:456-460 (1997). Antigenloading of dendritic cells may be achieved by incubating dendritic cellsor progenitor cells with the polypeptide, DNA (naked or within a plasmidvector) or RNA; or with antigen-expressing recombinant bacterium orviruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).Prior to loading, the polypeptide may be covalently conjugated to animmunological partner that provides T cell help (e.g., a carriermolecule). Alternatively, a dendritic cell may be pulsed with anon-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Vaccines and pharmaceutical compositions may be presented in unit-doseor multi-dose containers, such as sealed ampoules or vials. Suchcontainers are preferably hermetically sealed to preserve sterility ofthe formulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Preparation of Mtb72f (No His Tag) (SEQ ID No: 6) Constructionof the Mtb72f Expression Vector

Mtb72f is a fusion protein formed from 2 Mycobacterium tuberculosisproteins Mtb32 and Mtb39. Mtb72f is constructed by fusing Mtb39 and theN and C terminal portions of Mtb32 as follows: Mtb32 C-terminalend-Mtb39-Mtb32 N-terminal end. Specifically, Mtb72f protein wasgenerated by the sequential linkage in tandem of the open reading frames(ORFs) encoding the ˜14-kDa C-terminal fragment of Mtb32 (residues192-323; 132 amino acids) to the full length ORF of Mtb39 followed atthe C-terminus with the ˜20-kDa N-terminal portion (residues 1-195) ofMtb32. This was accomplished by using sequence-specific oligonucleotidescontaining unique restriction sites (EcoRI and EcoRV) and devoid of thestop codons at the C-terminal ends (in the case of Mtb32-C and Mtb39)for polymerase chain reaction (PCR) off of genomic DNA from the M.tuberculosis strain H37Rv.

The details of the process are as follows:

First, the DNA encoding the C-terminal portion of Mtb32 (Mtb32C) wascloned from H37Rv using PCR with the following oligonucleotides: 5′(5′-CAA-TTA-CAT-ATG-CAT-CAC-CAT-CAC-CAT-CAC-ACG-GCC-GCG-TCC-GAT-AAC-TTC-3′)and 3′ (5′-CTA-ATC-GAA-TCC-GGC-CGG-GGG-TCC-CTC-GGC-CAA-3′). The 5′oligonucleotide contained an NdeI restriction site (underlined)encompassing the ATG initiation codon. The 3′ oligonucleotide containedan EcoRI restriction site (underlined). These oligonucleotides were usedto amplify Mtb32C, a 396 nucleotide portion of Mtb32 and the resultingproduct was subcloned into the NdeI and EcoRI sites of an expressionvector. Digesting with EcoRI and EcoRV subsequently linearized theMtb32C plasmid.

For Mtb39, the following oligonucleotides were used for PCRamplification and cloning:5′-(5′-CTA-ATC-GAA-TTC-ATG-GTG-GAT-TTC-GGG-GCG-TTA-3′) and 3′(5′-CTA-ATC-GAT-ATC-GCC-GGC-TGC-CGG-AGA-ATG-CGG-3′). The 5′oligonucleotide contained an EcoRI restriction site (underlined) whilethe 3′ oligonucleotide contained an EcoRV restriction site (underlined).The full-length coding sequence of Mtb39 was amplified, digested, andsub-cloned in-frame downstream of Mtb32c using the predigested plasmidfrom the first step.

The 5′ and 3′ oligonucleotides of the N-terminal fragment of Mtb32 weredesigned as follows:5′-(5′-CTA-ATC-GAT-ATC-GCC-CCG-CCG-GCC-TTG-TCG-CAG-GAC-3′) and 3′(5′-CTA-ATC-GAT-ATC-CTA-GGA-CGC-GGC-CGT-GTT-CAT-AC-3′). Both sets ofoligonucleotides contained an EcoRV restriction site (underlined) whilethe 3′ oligonucleotide also included a stop codon (italics). Theoligonucleotides were designed to amplify a 585 bp portion of Mtb32encoding the predicted N-terminal domain of this protein. The resultingPCR product was sub-cloned into the Mtb32c-Mtb39 fusion plasmid. Theproper orientation of inserts and the absence of mutations was thenverified by DNA sequencing. For the final construct, used for making theMaster Cell Bank and Manufacturer's Working Cell Bank, the 6×Hisaffinity tag was removed by PCR and the open reading frame (ORF) forMtb72f was subcloned into pPDM, a pET derived expression vector. The ORFcodes for a polyprotein of about 72 kDa (Mtb72f), with domains organizedin the linear order: Mtb32C-Mtb39-Mtb32N. This DNA was then transformedinto the HMS174 pLysS strain of E. coli and used for testing, cellbanking, and manufacture.

Production of Mtb72f Bulk Drug Substance

The manufacturing process for the production of Mtb72f is summarized asfollows:

-   -   Fermentation followed by cell harvest by centrifugation, cell        disruption (microfluidizer) and centrifugation to yield an        inclusion body pellet;    -   Purification of the inclusion body pellet by extraction in 8M        urea, followed by Q Sepharose Fast Flow (QFF) chromatography,        Ceramic Hydroxyapatite (CHT) chromatography, diafilitration, and        sterilizing filtration to yield the purified bulk drug        substance.

Fermentation

Fermentations are performed at a 10 L working volume. The fermentor isinoculated with 300 mL of a shake flask culture of the working seedcells grown at 37° C. overnight. Both the inoculum and the fermentationuse a semidefined medium with plant-derived glycerol as the primarycarbon source. The composition of the medium is shown in the tablebelow. All medium components are sterilized by heating at 121° C. for 20minutes or by sterilizing filtration. During the fermentation thefermentor is maintained at a temperature of 37° C. Air is sparged at arate of 5 standard liters per minute (SLPM). The pH of the medium ismaintained at 7.0 by automatic addition of acid (H₂SO₄) or base (NaOH).The fermentor is programmed to control the dissolved oxygen at 30% byautomatically adjusting the agitation, while maintaining a minimumagitation of 200 rpm. Foam control within the fermentor is achieved bythe automatic addition of 1.05% SAG-471 silicone antifoam (Witco Corp.).When the cell density reaches an optical density (600 nm) ofapproximately 3.5, isopropyl-beta-D-thiogalactopyranoside (IPTG) isadded to the fermentor to a concentration of 1.0 mM. The IPTG inducesexpression of the recombinant gene encoding the Mtb72f protein. At 3.0hours post-induction, the fermentor is cooled and the cells areharvested by centrifugation in 1 L centrifuge bottles.

Composition of Fermentation Medium

Material Concentration Yeast Extract 15 g/L Glycerol 30 g/L Magnesiumsulfate, heptahydrate (MgSO₄— 0.5 g/L Potassium phosphate, monobasic(KH₂PO₄) 2.4 g/L Sodium phosphate, dibasic (Na₂HPO₄) 3.2 g/L Ammoniumchloride (NH4CI) 1.0 g/L Sodium chloride (NaCl) 0.5 g/L Kanamycinsulphate 30 mg/L Chloramphenicol 34 mg/L SAG-471 silicone antifoam(Witco Corp.) 0.0005% (v/v) (not included in

indicates data missing or illegible when filed

Isolation of Inclusion Bodies

The cell pellets are resuspended and pooled in 2.3 L of lysis Buffer (50mM NaCl, 10 mM Tris pH 8.0), and a M-1 10Y Microfluidizer® is used todisrupt the cells. The cells are passed through the Microfluidizer fivetimes at a pressure of 11,000±1,000 psi. The suspension is centrifugedat 8000×g in 500 mL bottles. Under these conditions, the inclusionbodies (IB) containing the Mtb72f protein are pelleted, while most ofthe cell debris remains in the supernatant. The IB pellets areresuspended in Wash Buffer (2 M urea, 50 mM NaCl, 10 mM Tris pH 8.0),followed by centrifugation at 8,000g. The supernatant fractions arediscarded and the IB pellets are stored at −70° C. to −80° C. untilneeded for further purification.

Purification of Polyprotein

The frozen IB preparations are thawed at 37° C. for 15 minutes and thenresuspended in 8 M urea, 50 mM NaCl, 20 mM Bis-tris propane, pH 7.0(Buffer A) using gentle mechanical agitation. The resuspended IBs arethen stirred at room temperature with a magnetic stir bar at 300 rpm for2 hrs. The IB extract is then centrifuged at high speed and theresultant supernatant fraction is filtered through a 0.45 uM filter(Pall, Supor) prior to chromatographic fractionation.

The IB extract is applied to a column containing Q Sepharose Fast Flow(QFF) anion exchange resin (10×12.5 cm Amersham/Pharmacia BPG; 1 Lpacked bed) previously sanitized with 1 N NaOH and then equilibratedwith Buffer A. The column is developed at a linear flow rate of 60 cm/hrwith Buffer A and the flow-through containing predominantly lower masscontaminants is collected for reference. The bulk of the Mtb72f iseluted in a single step using 8 M urea, 90 mM NaCl, 20 mM Bis-trispropane, pH 7.0 and is collected as a single bulk peak based onabsorbance.

QFF resins are highly cross-linked agarose resins with a quaternaryamine functional group that is positively charged in the conditions usedduring purification. The charged matrix allows for the binding ofvarious anions that can then be selectively eluted using a saltgradient. This anion exchange chromatography is used to separate nucleicacids and endotoxin, which bind tightly to the resin, from the protein,which is bound more weakly and elutes prior to these contaminants.Additionally, this step removes uncharged contaminants and a large partof the protein impurities.

The 90 mM NaCl eluate peak is from the QFF column is applied to a column(2.6×12 cm Amersham/Pharmacia XK26/20; 63 mL packed bed) containingMacroPrep® ceramic hydroxyapatite (CHT) (type I, 40 uM, BioRad)previously sanitized using 1 N NaOH and then equilibrated with Buffer C(8 M urea, 250 mM NaCl, and 20 mM Bis-tris propane, pH 7.0). Theflow-through material (FT1) containing the majority of the Mtb72f, freeof contaminants, is collected. The column is washed with Buffer C andany resultant UV-absorbing material is collected. Finally, the column iseluted in Buffer D (8 M urea, 200 mM sodium phosphate, pH 7.4).

MacroPrep® CHT is a spherical, macroporous form of hydroxyapatite[Ca₅(PO₄)₃OH]₂. CHT chromatography can be a highly selective method ofpurification if the proper binding and elution conditions are found. Themodes of binding include ion exchange type binding to charged calciumand phosphate ions as well as chelation of molecules. DNA will bind tothis resin and high selectivity for individual proteins can be achieved.The conditions used for the purification of Mtb72f serve as a polishingstep allowing virtually complete removal of detectable host cellcontaminants.

During chromatographic separations, ultraviolet (UV) absorbance,conductivity, pressure, pH, flow-rate, and ambient temperature aremonitored and recorded. The initial CHT flow-through material (FT1) isused for further downstream processing.

Diafiltration and Sterile Filtration

Diafiltration is performed on the CHT FT1 pool to remove the urea andreplace the buffer with 20 mM Tris pH 7.5. The diafiltration isperformed using a Pall Minim™ system with an LV-Centramate™ tangentialflow filtration device with a 30 kDa molecular weight cutoff (MWCO)ultrafiltration membrane. The Mtb72f solution in 20 mM Tris pH 7.5 isfilter sterilized using a 0.2-um sterilizing filter (Millipak 40). FiftymL of the solution are distributed into sterile 60 mL PETG (polyethyleneterephthalate copolymer) media bottles, then frozen and stored at −70°C. This material is the Mtb72f purified bulk drug substance.

Example 2 Preparation of Mtb72f (6 His Tag) (SEQ ID No: 2)

The method of Example 1 may be followed, except that the step ofsubcloning into pPDM in order to remove the His Tag is omitted.

Example 3 Preparation of M72 (2 His Tag) (SEQ ID No: 4) Construction ofthe M72 Expression Vector

Starting material for the construction of M72 antigen was therecombinant plasmid 6His-Mtb72fmut. 6His-Mtb72fmut was prepared bysite-directed mutagenesis using the 6his-Mtb72f recombinant plasmid (seeExample 1) as template. Site-directed mutagenesis involved replacing thecodon for Ser at position 710 in SEQ ID No: 1 with a codon for Ala. Thedeletion of four N-terminal histidines present on the 6His-Mtb72fmutconstruct (Corixa plasmid) was achieved with “Gene Tailor Site-DirectedMutagenesis System” (Invitrogen), leading to the expected 2His-Mtb72Fmutconstruct. After sequence verification, 2His-Mtb72fmut coding sequencewas excized from the plasmid (by enzymatic restriction), gel purifiedand ligated into pET29a expression vector resulting in the finalrecombinant plasmid pET29a/2His-Mtb72fmut. After sequence verificationthe recombinant plasmid was given the official designation pRIT15497 andused to transform HMS174(DE3) host cells. pRIT15497 codes for a 725amino-acid protein named M72.

Production of M72 Protein

The same production process as described for Mtb72f (see Example 1) maybe employed., except that for M72 production, chloramphenicol is absentin the fermentation medium.

Biological Example 1 A Mouse Model of an Inactive/Latent State of M.tuberculosis Infection

To establish a mouse model of latent M. tuberculosis infection, the SWRstrain was used. SWR mice are not immunocompromised, but are deficientfor secretion of complement component C5 (see, Ooi and Colten, Nature(1979) 282:207-8). SWR mice are incapable of establishing a chronicstate of Mtb infection, but develop diffuse granulomatous pneumoniacharacterized by large epitheloid and foamy macrophages with crystalloidinclusions (neutrophil or eosinophil-derived granules that have beenphagocytosed), multifocal necrosis, neutrophil accumulation and scantlymphocytes (see, Turner, et al., J Submicrosc Cytol Pathol. (2001)33(1-2):217-9; and Turner, et al., Infect Immun. (2003) 71(9):5266-72).Following is the protocol for using the Swiss Webster (SWR/J) mousestrain in a model of latent M. tuberculosis infection to evaluate thetherapeutic efficacy of Mtb72f (SEQ ID No:6) formulated with AS01Badjuvant. Double strength AS01B is prepared by adding QS21 (5 μg) tosmall unilamellar vesicles (SUV) of dioleoyl phosphatidylcholine (100μg) containing cholesterol (25 μg) (WO 96/33739) and monophosphoryllipid A (MPL) (5 μg) in the membrane (see, U.S. Patent Publication No.2003/0143240). An aliquot for injection (50 μl) is prepared by mixing 4μg of protein in buffer (PBS pH 6.8) with 50 μl of double strengthAS01B. Each mouse received two injections of 50 μl (i.e. 8 μg ofprotein).

A representative timeline for establishing a model of a latent M.tuberculosis infection is schematically depicted in FIG. 1.

Day 1: Infect via aerosol with 50-100 colony forming units (CFU) M.tuberculosis organismsDay 30-90: Treat a subset of mice with 50 mg rifampin/85 mg isoniazideper Liter of drinking waterDay 61: All mice receiving the candidate vaccine 5 should be immunizedwith rMtb72f+AS01BDay 82: All mice receiving the candidate vaccine should be immunizedwith rMtb72f+AS01BDay 103: All mice receiving the candidate vaccine should be immunizedwith rMtb72f+AS01BDay 113: Bleed for IgG assaysVarious Timepoints: Take spleens and lungs for CFU enumeration &immunogenicityVariation 1→Treat with chemotherapy for 60 days. Starting at day 30→Restfor 3, 4, 5 months→CFU in 2 mice at each time point and leave 4-7 micefor survival studiesVariation 2→Treat with chemotherapy for 90 days. Starting at day 30→Restfor 4, 5 months→CFU in 2 mice at each time point and leave 7 mice forsurvival studiesVariation 3→Rest for 4, 5, 6 months→CFU in 2 mice at each time point andleave 4 mice for survival studiesVariation 4→Treat with chemotherapy for 60 days. Starting at day 30→3immunizations with r72F+AS01B intramuscularly (i.m.) starting at Day60→Rest for 3, 4, 5 months→CFU in 2 mice at each time point and leave4-7 mice for survival studiesVariation 5→Treat with chemotherapy for 90 days. Starting at day 30→3immunizations with r72F+AS01B i.m starting at Day 60→Rest for 4, 5months→CFU in 2 mice at each time point and leave 4-7 mice for survivalstudies

Analysis of post-infection antibody responses using rMtb72f to coat theELISA plates revealed that those groups that received a combination ofchemotherapy and Mtb72f+AS01B immunization had a higher antibodyresponse (OD up to 2.0) than mice that were untreated or receivedchemotherapy treatment alone (OD of less than 0.5) (FIG. 2). Miceimmunized with Mtb72f mounted a sizeable Mtb72f-specific antibodyresponse (OD of between 1.5 and 2.5) whether they received 60 or 90 daysof chemotherapy (FIG. 3).

Spleen cells were harvested from mice at various intervals after themice were infected with M. tuberculosis. The splenocytes werere-stimulated in vitro with recombinant antigens to measure IFN-γsecretion. IFN-γ levels produced by these cells were uniformlynegligible in groups 1 (untreated) and 2 (chemotherapy only) at day 60,with the exception of Mtb39. Positive control stimulations with conA,PPD and BCG lysate demonstrated the cells were capable of synthesizingand secreting IFN-γ in response to other stimulatory molecules (FIG. 4).IFN-γ levels were high in groups receiving Mtb72f+AS01B, but they werelow or negligible in groups that had not been immunized withMtb72f+AS01B, whether or not they had received chemotherapy (FIG. 5).

During the course of tuberculosis infection and subsequent treatment,specific T cells respond. Using intracellular cytokine staining forIFN-γ the percentage of specific CD4⁺ cell responses to Mtb72F wasmeasured (FIG. 6). There seemed to be no change in the Mtb72F specificCD4⁺ IFN γ⁺ T cell responses during the course of chemotherapy alone atany time point as measured by this assay (FIG. 7). At day 120 post Mtbinfection, the trend of CD4⁺ IFNγ⁺ response to Mtb72F in groupsreceiving the Mtb72f plus AS01B vaccine, appeared to increase with thelength of time on chemotherapy (FIG. 7).

The results of our experiments demonstrate that SWR mice are susceptibleto infection with M. tuberculosis. If left untreated, SWR mice die by115 days post Mtb infection (FIGS. 8 and 9). Mean survival time for micereceiving 60 days of combination chemotherapy was 170 days (FIGS. 8 and9). Mean survival time for mice receiving 60 days of combinationchemotherapy and 3 immunizations of Mtb72f/AS01B was 215 days (FIGS. 8and 9). Survival for the group of mice receiving chemotherapy issignificantly different (95% confidence interval (p=0.0067) from thosereceiving chemotherapy and the Mtb72f/AS01B vaccine.

1-31. (canceled)
 32. A method for treating a subject infected withMycobacterium tuberculosis comprising administering to a subjectinfected with M. tuberculosis (a) a pharmaceutical compositioncomprising an immunologically effective amount of: (i) a Mtb72f fusionprotein or an immunogenic fragment thereof from a Mycobacterium speciesof the tuberculosis complex and an adjuvant; or (ii) a nucleic acidencoding a Mtb72f fusion protein or an immunogenic fragment thereof froma Mycobacterium species of the tuberculosis complex, and (b) one or morechemotherapeutic agents effective against a M. tuberculosis infectionwherein the Mtb72f fusion protein or immunogenic fragment thereof of (i)or encoded by the nucleic acid of (ii) induces an immune responseagainst M. tuberculosis in the subject.
 33. The method of claim 32,wherein the pharmaceutical composition of (a) comprises a Mtb72f fusionprotein or an immunogenic fragment thereof from a Mycobacterium speciesof the tuberculosis complex and an adjuvant.
 34. The method of claim 32,wherein the pharmaceutical composition comprises a nucleic acid encodinga Mtb72f fusion protein or an immunogenic fragment thereof from aMycobacterium species of the tuberculosis complex.
 35. The method ofclaim 32, wherein the Mtb72f fusion protein is from Mycobacteriumtuberculosis.
 36. The method of claim 35, wherein the Mtb72f fusionprotein is a polypeptide comprising residues 8-729 of SEQ ID NO:2. 37.The method of claim 35, wherein the Mtb72f fusion protein is apolypeptide comprising residues 4-725 of SEQ ID NO:4.
 38. The method ofclaim 33, wherein the adjuvant is selected from the group consisting of(i) 3D-MPL and QS21 in a liposome formulation, and (ii) 3D-MPL and QS21and an oil in water emulsion.
 39. The method of claim 34, wherein thenucleic acid comprises nucleotides 63-2222 of SEQ ID NO:1.
 40. Themethod of claim 34, wherein the nucleic acid comprises nucleotides10-2175 of SEQ ID NO:3.
 41. The method of claim 34, wherein the nucleicacid is administered in an adenovirus vector.
 42. The method of claim34, wherein the nucleic acid is administered in a mutant Mycobacteriumor Bacillus host cell vector.
 43. The method of claim 32, wherein theone or more chemotherapeutic agents effective against a M. tuberculosisinfection of (b) is administered over a period of time.
 44. The methodof claim 43, wherein the one or more chemotherapeutic agents effectiveagainst a M. tuberculosis infection of (b) is administered over about 1week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about3 months, about 4 months, about 5 months, about 6 months, about 8 monthsor about 1 year.
 44. The method of claim 32, wherein the one or morechemotherapeutic agents is selected from isoniazid and rifampin.
 45. Themethod of claim 32, wherein the subject is first administered said oneor more chemotherapeutic agents of (b) and then administered saidpharmaceutical composition of (a).
 46. The method of claim 32, whereinthe subject is first administered said pharmaceutical composition of(a), and then administered said one or more chemotherapeutic agents of(b).
 47. The method of claim 32, wherein administration of the one ormore chemotherapeutic agents of (b) and the pharmaceutical compositionof (b) is initiated concurrently.
 48. A method for reducing the timecourse of chemotherapy for treating a subject infected withMycobacterium tuberculosis, the method comprising administering to asubject infected with M. tuberculosis (a) a pharmaceutical compositioncomprising an immunologically effective amount of: (i) a Mtb72f fusionprotein or an immunogenic fragment thereof from a Mycobacterium speciesof the tuberculosis complex and an adjuvant; or (ii) a nucleic acidencoding a Mtb72f fusion protein or an immunogenic fragment thereof froma Mycobacterium species of the tuberculosis complex, and (b) one or morechemotherapeutic agents effective against a M. tuberculosis infectionwherein the Mtb72f fusion protein or immunogenic fragment thereof whichis administered in (i) or encoded by the nucleic acid of (ii) induces animmune response against M. tuberculosis in the subject thereby allowingfor reducing the time course of chemotherapy against a M. tuberculosisinfection.